Presently there are four different classes of enzymes are known to have at least one enzyme member can hydrolyze phytate and release phosphorus. These four classes of phosphatase enzymes are histidine acid phosphatase (HAP), β-Propeller phytase (BPP), protein tyrosine phosphatase (PTP), and purple acid phosphatase (PAP). Out of these four classes, only members of the HAP have been identified has having high specific activity and other features necessary to be marketed commercially. Additionally, only a few HAPs utilize phytic acid effectively as a substrate (Mullaney et al., Inositol phosphates: Linking Agriculture and the Environment 2007, 97-110, Eds B. Turner, A. Richardson, and E Mullaney CABI; Nakaskima et al., Microbial Ecology, (2006) 53:82-88).
Phytase enzymes are a group of histidine acid phosphatases (HAP) with great potential for improving mineral nutrition and protecting the environment from phosphorus pollution coming from animal waste (Lei et al., J. Appl. Anim. Res., 17:97-112 (2000)). Aspergillus niger NRRL 3135 phyA phytase has been cloned (Mullaney et al., “Positive Identification of a Lambda gt11 Clone Containing a Region of Fungal Phytase Gene by Immunoprobe and Sequence Verification,” Appl. Microbiol. Biotechnol. 35:611-614 (1991); and Van Hartingsveldt et al., “Cloning, Characterization and Overexpression of the Phytase-Encoding Gene (phyA) of Aspergillus niger,” Gene 127:87-94 (1993)) and overexpressed for commercial use as animal feed additive (Van Dijck, J. Biotechnology 67:77-80 (1999)). Recent information on its molecular structure from its X-ray-deduced three dimensional structure (Kostrewa et al., “Crystal Structure of Phytase from Aspergillus ficuum at 2.5 Å Resolution,” Nat. Struct. Biol. 4:185-190 (1997)) has facilitated several studies to enhance the specific activity of other phytases. Analysis of available 3-D structure models of fungal histidine acid phosphatases reveals that disulfide bridges allow for the convergence of distant regions of the amino acid sequence to obtain the required molecular folding of the molecule for specifically function notably at the active site and substrate-binding domain.
Phytase from Aspergillus fumigatus has been studied for its superior thermotolerance properties, significant levels of activity over a wide range of pH, and resistance to hydrolysis by pepsin (Pasamontes et al., “Gene Cloning, Purification, and Characterization of a Heat-Stable Phytase From the Fungus Aspergillus fumigatus,” Appl. Environ. Microbiol. 63:1696-1700 (1997); and Rodriguez et al., “Expression of the Aspergillus fumigatus Phytase Gene in Pichia pastoris and Characterization of the Recombinant Enzyme,” Biochem. Biophys. Res. Commun. 268:373-378 (2000)). However, specific activity of this phytase is not as high as some other fungal phytases such as those produced by A. terreus or A. niger. As disclosed in Wodzinski, et al., 1996, Adv Appl Microbiol., 42:263-302, wild type strain A. niger NRRL 3135 produced the highest yield and activity of phytase. As such, there is a need to further develop an A. niger phytase for maintaining activity upon pelletizing temperatures. Ideally, the phytase would be incorporated into animal feed in feed to monogastric animals in a pelletized form.
Given the higher specific activity of A. niger phytase, there is a need to explore the role of the disulfide bridge for structural folding and the effect on biological functions. To date, there has been no disclosure reported on the effect of removal of any disulfide bonds in fungal HAPhys.
Studies have reported on the removal of disulfide bridges in Escherichia coli HAPhys. Specifically, E. coli phytase, AppA, has four disulfide bridges (Lim et al., 2000). Rodriguez et al., Archives of Biochemistry and Biophysics, Volume 382, Issue 1, Pages 105-112 (2000) reported the replacement of one cysteine that was involved in forming a disulfide bridge in E. coli phytase, coupled with replacement of other targeted amino acids yielded a phytase with improved thermostability and catalytic efficiency. In another study to define the function of a protein-disulfide isomerase, DsbC, on E. coli phytase, a series of phytase mutants lacking either one or both of the cysteines for each of the four disulfide bonds was generated and the phytase produced was characterized (Berkman et al., 2005). While all single E. coli mutant phytases displayed lower levels of acid phosphatase activity than the wild type (WT) phytase, one double mutant, C200S C210S, in that study did have higher activity. The degree of loss of activity varied with the cysteine(s) replaced and the results confirmed a relationship between disulfide bridges and catalytic function of the enzyme.
The native NRRL 3135 phyA phytase is a stable enzyme (Ullah et al., “Extracellular Phytase (E. C. 3.1.3.8) from Aspergillus niger NRRL 3135: Purification and Characterization,” Prep. Boichem. 17:63-91 (1987)) that has a high specific activity for phytic acid (Wyss et al., “Biochemical Characterization of Fungal Phytases (Myo-inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties,” Applied and Envir. Micro. 65:367-373 (1999)). This has contributed to its acceptance by the animal feed industry (Wodzinski et al., “Phytase,” Advances in Applied Microbiology 42:263-302 (1996)). It has also been widely researched and utilized to engineer improved features into other fungal phytases by recombinant DNA techniques (Wyss et al., “Biophysical Characterization of Fungal Phytases (Myo-iositol Hexakisphosphate Phosphohydrolases): Molecular Size, Glycosylation Pattern, and Engineering of Proteolytic Resistance,” Applied and Envir. Micro. 65:359-366 (1999); and Lehmann et al., “Exchanging the Active Site Between Phytases for Altering the Functional Properties of the Enzyme,” Protein Science 9:1866-1872 (2000)).
NRRL 3135 PhyA is known to have an active site motif characteristic of the histidine acid phosphatase (HAP) class of enzymes (Ullah et al., “Cyclohexanedione Modification of Arginine at the Active Site of Aspergillus ficuum Phytase,” Biochem. Biophys. Res. Commun. 178:45-53 (1991); and Van Etten et al., “Covalent Structure, Disulfide Bonding, and Identification of Reactive Surface and Active Site Residues of Human Prostatic Acid Phosphatase,” J. Biol. Chem. 266:2313-2319 (1991)). Previous studies of the crystal structure of the A. niger NRRL 3135 phyA (Kostrewa et al., “Crystal. Structure of Phytase from Aspergillus ficuum at 2.5 Å Resolution,” Nat. Struct. Biol. 4:185-190 (1997)) and phyB (Kostrewa et al., “Crystal Structure of Aspergillus niger pH 2.5 Acid Phosphatase at 2.4 Å Resolution,” J. Mol. Biol. 288:965-974 (1999)) molecules have provided researchers with structural models of both these enzymes. These models have facilitated the identification of the residues constituting the catalytic active center of the molecules, i.e., both the active site and substrate specificity site. Its active site consists of a catalytic center (R81, H82, R66, R156, H361 D362) (Mullaney et al., (2000) Advances in Applied Microbiology 47:157-199) and a substrate specificity site (K91, K94, E228, D262, K300, K301) (Kostrewa et al., (1999) “Crystal Structure of Aspergillus niger pH 2.5 Acid Phosphatase at 2.4 Å Resolution,” J. Mol. Biol., 288:965-974). The amino acid numbers refer to full length phytase encoded by the A. niger NRRL 3135 phyA gene (NCBI Accession No. AAA32705). Amino acid reference numbers in Kostrewa et al., “Crystal Structure of Aspergillus niger pH 2.5 Acid Phosphatase at 2.4 Å Resolution,” J. Mol. Biol. 288:965-974 (1999) were derived from a slightly truncated sequence. The narrow substrate specificity and the unique pH activity profile of this phytase, a drop in activity in the pH range 3.0-5.0; have been ascribed to the interaction of these acidic and basic amino acids comprising the substrate specificity site. This low activity at this intermediate pH range is not observed in other fungal phytases and is an undesirable feature of A. niger NRRL 3135 phyA.
Phytate (myo-inositol hexakisphosphate) is the major form of phosphorus in plant origin feed. Non-ruminants such as poultry and swine are unable to utilize phytate phosphorus in soy-corn based diet. Supplemental microbial phytase has been used successfully to improve phytate phosphorus utilization and to reduce phosphorus excretion by these animals (Lei et al., “Supplementing Corn-Soybean Meal Diets with Microbial Phytase Linearly Improves Phytate Phosphorus Utilization by Weanling Pigs,” J. Anim. Sci. 71:3359-3367 (1993); and Lei et al., “Supplemental Microbial Phytase Improves Bioavailability of Dietary Zinc to Weanling Pigs,” J. Nutr. 123:1117-23 (1993)). The most widely used commercial phytase is Aspergillus niger PhyA is used as a feed additive. However, PhyA has a unique pH profile of having two pH optima ranges of between 5 to 5.5 and 2.5. A drop in activity exists in the range of pH 3 to 5 with another depression at pH 3.5. Phytate degradation by dietary phytase takes place mainly in the stomach in Sus scrofa scrofa (Yi et al., “Sites of Phytase Activity in the Gastrointestinal Tract of Young Pigs,” Animal Feed Science Technology 61:361-368 (1996)). Inasmuch as Sus scrofa scrofa stomach has pH ranges from 2.5 to 3.5 and there is a depression in PhyA activity at a pH of 3.5 resulting in compromising unmodified PhyA's efficacy for phytase activity. As such there is a need in the art to optimize phytase activity at a range optimal for monogastric animals for in vivo phytate degradation.
Additionally, feed formulations for monogastric animals containing PhyA is heated during a pelletization process. The heat in the pelletization process denatures the enzyme and significantly lowers the activity of the enzyme. Typically, pelletization via a heated extrusion process exposes the feed material to a temperature of approximately 70° C. to 90° C. for a period up to five minutes. As such, there is a need to generate a phytase as an animal feed additive such that the enzyme is heat tolerant and can withstand irreversibly denaturation upon exposure to elevated temperatures.